Conventional diagnosis of microbial infections generally relies on cell culturing to detect and identify the microorganism responsible for the infection. While cell culturing is inexpensive, it can be relatively slow because it relies on visual detection of individual bacterial colonies (i.e., ˜106 bacteria). For example, while colonies of a fast growing bacterium can be observed between ˜24 to 48 hours, slower growing bacteria require incubation periods of a week or more to detect bacterial colonies. As further alternatives, instruments have been developed using various principles of detection including infrared or fluorescence spectroscopy, bioluminescence, and flow cytometry (Basile, et al. (1998) Trends Anal. Chem. 17:95-109; Bird, et al. (1989) Rapid Salmonella Detection by a Combination of Conductance and Immunological Techniques; Blackwell Sci. Publications: Oxford, Vol. 25; Fenselau, Ed. (1994) Mass Spectrometry for the Characterization of Microorganisms Washington D.C., Vol. 240; Lloyd, Ed. (1993) Flow Cytometry in Microbiology; Springer-Verlag London Limited: Germany; Perez, et al. (1998) Anal. Chem. 70:2380-2386; Wyatt (1995) Food Agri. Immunol. 7:55-65). Among these, the primary physical/chemical methods of bacterial detection are those which involve the detection of some naturally occurring component of the bacterium.
BACT/ALERT uses, for example, a colorimetric sensor detection system which detects microorganism growth by the production of CO2. When the CO2 levels reach a certain level, the sensor turns yellow giving a positive result for bacteria present. This system can be used for a wide variety of microorganisms and has a success rate of 95% in 24 hours and 98% in 72 hours (Weinstein, et al. (1995) J. Clin. Microbiol. 33:978-981; Wilson, et al. (1995) J. Microbiol. 33:2265-2270; Wilson, et al. (1992) J. Clin. Microbiol. 30:323-329).
Other devices and methods for detecting microorganisms are provided in U.S. Pat. Nos. 5,094,955; 6,777,226; 6,197,577; 5,976,827; and 5,912,115. In general, these devices rely on the use of a single sensor (e.g., pH or carbon dioxide indicator) in a layer adjacent to a layer of growth medium for detecting the presence of a bacterium.
Bacterial identification methods usually include a morphological evaluation of microorganisms as well as tests for the organism's ability to grow in various media sources under various conditions. These techniques allow for the detection of single organisms, however, amplification of the signal is required through growth of a single cell into a colony and no single test provides a definitive identification of an unknown bacterium. Traditional methods for the identification of bacteria involve pre-enrichment, selective enrichment, biochemical screening, and serological confirmation (Tietjen & Fung (1995) Crit. Rev. Microb. 21:53-83; Kaspar & Tartera (1990) Methods Microbiol. 22:497-530; Helrich (1990) Official Methods of Analysis of Association of Official Analytical Chemists; 15 ed.; AOAC: Arlington, Va., Vol. 2; Hobson, et al. (1996) Biosensors & Bioelectronics 11:455-477).
Alternative methods such as immunoassays and PCR-based approaches have been pursued with varying degrees of success (Iqbal, et al. (2000) Biosensors & Bioelectronics 15:549-578; Morse (2000) Detecting Biological Warfare Agents; Lynne Rienner Publishers, Inc: Boulder, Colo.). However, in the case of PCR, such an approach is expensive and requires pure samples, hours of processing, and an expertise in microbiology (Spreveslage, et al. (1996) J. Microbiol. Methods 26:219-224; Meng, et al. (1996) Intl. J. Food Microbiol. 32:103-113). An alternative method, gas chromatography/mass spectrometry (GC/MS), has been used to produce a fatty acid profile or “fingerprint” for the detection and identification of microorganisms (Swaminathan & Feng (1994) Ann. Rev. Microbiol. 48:401-426).
Array-based vapor sensing is an approach toward the detection of chemically diverse analytes. Based on cross-responsive sensor elements, rather than specific receptors for specific analytes, these systems produce composite responses unique to an odorant in a fashion similar to the mammalian olfactory system (Stetter & Pensrose, Eds. (2001) Artificial Chemical Sensing: Olfaction and the Electronic Nose; Electrochem. Soc.: New Jersey; Gardner & Bartlett (1999) Electronic Noses: Principles and Applications; Oxford University Press: New York; Persuad & Dodd (1982) Nature 299:352; Albert, et al. (2000) Chem. Rev. 100:2595-2626; Lewis (2004) Acc. Chem. Res. 37:663-672; James, et al. (2005) Microchim. Acta 149:1-17; Walt (2005) Anal. Chem. 77:45A). In such arrays, one receptor responds to many analytes and many receptors respond to any given analyte. A distinct pattern of responses produced by the calorimetric sensor array provides a characteristic fingerprint for each analyte. Using such systems, volatile organic compounds have been detected and differentiated (Rakow & Suslick (2000) Nature 406:710-713; Suslick & Rakow (2001) Artificial Chemical Sensing: Olfaction and the Electronic Nose; Stetter & Penrose, Eds.; Electrochem. Soc.: Pennington, N.J.: pp. 8-14; Suslick, et al. (2004) Tetrahedron 60:11133-11138; Suslick (2004) MRS Bulletin 29:720-725; Rakow, et al. (2005) Angew. Chem. Int. Ed. 44:4528-4532; Zhang & Suslick (2005) J. Am. Chem. Soc. 127:11548-11549).
Array technologies of the prior art generally rely on multiple, cross-reactive sensors based primarily on changes in properties (e.g., mass, volume, conductivity) of some set of polymers or on electrochemical oxidations at a set of heated metal oxides. Specific examples include conductive polymers and polymer composites (Gallazzi, et al. (2003) Sens. Actuators B 88:178-189; Guadarrana, et al. (2002) Anal. Chim. Acta 455:41-47; Garcia-Guzman, et al. (2003) Sens. Actuators B 95:232-243; Burl, et al. (2001) Sens. Actuators B 72:149-159; Wang, et al. (2003) Chem. Mater. 15:375-377; Hopkins & Lewis (2001) Anal. Chem. 73:884-892; Feller & Grohens (2004) Sens. Actuators B 97:231-242; Ferreira, et al. (2003) Anal. Chem. 75:953-955; Riul, et al. (2004) Sens. Actuators B 98:77-82; Sotzing, et al. (2000) Anal. Chem. 72:3181-3190; Segal, et al. (2005) Sens. Actuators B 104:140-150; Burl, et al. (2002) Sens. Actuators B 87:130-149; Severin, et al. (2000) Anal. Chem. 72:658-668; Freund & Lewis (1995) Proc. Natl. Acad. Sci. U.S.A. 92:2652-2656; Gardner, et al. (1995) Sens. Actuators B 26:135-139; Bartlett, et al. (1989) Sens. Actuators B 19:125-140; Shurmer, et al. (1990) Sens. Actuators B 1:256-260; Lonergan, et al. (1996) Chem. Mater. 8:2298-2312), polymers impregnated with a solvatochromic dye or fluorophore (Chen & Chang (2004) Anal. Chem. 76:3727-3734; Hsieh & Zellers (2004) Anal. Chem. 76:1885-1895; Li, et al. (2003) Sens. Actuators B 92:73-80; Albert & Walt (2003) Anal. Chem. 75:4161-4167; Epstein, et al. (2002) Anal. Chem. 74:1836-1840; Albert, et al. (2001) Anal. Chem. 73:2501-2508; Stitzel, et al. (2001) Anal. Chem. 73:5266-5271; Albert & Walt (2000) Anal. Chem. 72:1947-1955; Dickinson, et al. (1996) Nature 382:697-700; Dickinson, et al. 1996) Anal. Chem. 68:2192-2198; Dickinson, et al. (1999) Anal. Chem. 71:2192-2198), mixed metal oxide sensors (Gardner & Bartlett (1992) Sensors and Sensory Systems for an Electronic Nose; Kluwer Academic Publishers: Dordrecht; Zampolli, et al. (2004) Sens. Actuators B 101:39-46; Tomchenko, et al. (2003) Sens. Actuators B 93:126-134; Nicolas & Romain (2004) Sens. Actuators B 99:384-392; Marquis & Vetelino (2001) Sens. Actuators B 77:100-110; Ehrmann, et al. (2000) Sens. Actuators B 65:247-249; Getino, et al. (1999) Sens. Actuators B 59:249-254; Heilig, et al. (1997) Sens. Actuators B 43:45-51; Gardner, et al. (1991) Sens. Actuators B 4:117-121; Gardner, et al. (1992) Sens. Actuators B 6:71-75; Corcoran, et al. (1993) Sens. Actuator B 15:32-37; Gardner, et al. (1995) Sens. Actuators B 26:135-139), and polymer coated surface acoustic wave (SAW) devices (Grate (2000) Chem. Rev. 100:2627-2648; Hsieh & Zellers (2004) Anal. Chem. 76:1885-1895; Grate, et al. (2003) Anal. Chim. Acta 490:169-184; Penza & Cassano (2003) Sens. Actuators B 89:269-284; Levit, et al. (2002) Sens. Actutors B 82:241-249; Grate, et al. (2001) Anal. Chem. 73:5247-5259; Hierlemann, et al. (2001) Anal. Chem. 73:3458-3466; Grate, et al. (2000) Anal. Chem. 72:2861-2868; Ballantine, et al. (1986) Anal. Chem. 58:3058-3066; Rose-Pehrsson, et al. (1988) Anal. Chem. 60:2801-2811; Patrash & Zellers (1993) Anal. Chem. 65:2055-2066). However, the sensors disclosed in these prior art references do not provide a diversity of interactions with analytes; interactions are limited to the weakest and least specific of intermolecular interactions, primarily van der Waals and physical adsorption interactions between sensor and analyte. As such, both sensitivity for detection of compounds at low concentrations relative to their vapor pressures and selectivity for discrimination between compounds is compromised with these prior art sensors.
Cross-responsive sensor technologies have also been applied to the identification of bacteria (Lai, et al. (2002) Laryngoscope 112:975-979; McEntegart, et al. (2000) Sensors and Actuators B 70:170-176; Gibson, et al. (1997) Sensors and Actuators B 44:413-422; Ivnitski, et al. (1999) Biosensors & Bioelectronics 14:599-624). These cross-responsive sensor technologies have employed a variety of chemical interaction strategies, including the use of conductive polymers (Freund & Lewis (1995) Proc. Natl. Acad. Sci. USA FIELD Publication Date 92:2652-2656), conductive polymer/carbon black composites (Lonergan, et al. (1996) Chem. Mater. 8:2298-2312), fluorescent dye/polymer systems (Walt (1998) Acc. Chem. Res. 31:267-278), tin oxide sensors (Heilig, et al. (1997) Sensors and Actuators, B: Chemical B43:45-51), and polymer-coated surface acoustic wave (SAW) devices (Grate (2000) Chem. Rev. 100:2627-2647). For example, an array of four metal oxide sensors has been used to detect and identify six pathogenic bacteria by sampling the headspace over the growing microorganisms, wherein the sensor correctly identified/classified 62% of the pathogens (Craven, et al. (1994) Neural Networks and Expert Systems in Medicine and Healthcare; University of Plymouth: Plymouth). The use of such technologies for medical application has been described (Thaler, et al. (2001) Am. J. Rhinology 15:291-295); however, these systems employ the detection of chemically non-coordinating organic vapors without exploring the detection of the most toxic and odiferous compounds (e.g., phosphines and thiols). In general, most cross-responsive sensor devices of the prior art have limited detection sensitivity and remain quite non-selective (O'Hara (2005) Clin. Microbiol. Rev. 18:147-162).
Additional devices for detecting microorganisms are disclosed in U.S. Pat. Nos. 6,030,828; 4,297,173; 4,264,728; 5,795,773; 5,856,175; 6,855,514; 7,183,073; and U.S. Patent Application No. 2005/0170497.
Needed is a cost-efficient, non-invasive, sensitive and selective sensor which can detect, quantify and discriminate between microorganisms. The present invention meets this long-felt need.